Multistate Redox-Switchable Ion Transport Using Chalcogen-Bonding Anionophores

Synthetic supramolecular transmembrane anionophores have emerged as promising anticancer chemotherapeutics. However, key to their targeted application is achieving spatiotemporally controlled activity. Herein, we report a series of chalcogen-bonding diaryl tellurium-based transporters in which their anion binding potency and anionophoric activity are controlled through reversible redox cycling between Te oxidation states. This unprecedented in situ reversible multistate switching allows for switching between ON and OFF anion transport and is crucially achieved with biomimetic chemical redox couples.


Materials and methods
All reagents and solvents were purchased from commercial sources and used without further purification. Lipids were purchased from Avanti polar lipids and used without further purification. Where necessary, solvents were dried by passing through an MBraun MPSP-800 column and degassed with nitrogen. Column chromatography was carried out on Merck® silica gel 60 under a positive pressure of nitrogen. Where mixtures of solvents were used, ratios are reported by volume. NMR spectra were recorded on a Bruker AVIII 400, Bruker AVII 500 (with He cryoprobe) and Bruker AVIIIHD 500 spectrometers. Chemical shifts are reported as δ values in ppm. Mass spectra were carried out on an Agilent 6120 bench-top single quadrupole, a Waters LCT Premier XE benchtop (oa-TOF) and a Thermo Exactive HighResolution Orbitrap FTMS spectrometer. Fluorescence spectroscopic data were recorded using a Horiba Duetta fluorescence spectrophotometer, equipped with Peltier temperature controller and stirrer. Experiments were conducted at 25°C unless otherwise stated. Vesicles were prepared as described below using Avestin "LiposoFast" extruder apparatus, equipped with polycarbonate membranes with 200 nm pores. GPC purification of vesicles was carried out using GE Healthcare PD-10 desalting columns prepacked with Sephadex G-25 medium
Transport assays with HPTS In a typical experiment, the LUVs containing HPTS (25 µL, final lipid concentration 31.3 µM) were added to buffer (1950 µL of 100 mM NaCl, 10 mM HEPES, pH 7.0) at 25°C under gentle stirring. A pulse of NaOH (20 µL, 0.5 M) was added to initiate the experiment, before the test transporter (various concentrations, in 5 µL DMSO) was added. Detergent (25 µL of Triton X-100 in 7:1 (v/v) H2O-DMSO) was added at the end of the run after a total of 276 s to calibrate the assay. The fluorescence emission was monitored at λem = 510 nm (λex = 405/460 nm). The fractional fluorescence intensity (Irel) was calculated from equation (S1), where Rt is the fluorescence ratio at time t, R0 is the fluorescence ratio prior to addition of transporter, and Rd is the fluorescence ratio after the addition of detergent.
The fractional fluorescence intensity (Irel) immediately prior to lysis, defined as the fractional activity y, was plotted as a function of the ionophore concentration (x / µM). Hill coefficients (n) and EC50 values were calculated by fitting to the Hill equation (S2): where y0 is the fractional activity in the absence of transporter, ymax is the fractional activity with excess transporter, x is the transporter concentration in the cuvette. Experiments with DPPC lipids were conducted in the same way. For elevated temperature studies, the buffer was equilibrated at 45°C (using the Peltier temperature controller) for 5 minutes prior to initiating the experiment.
Experiments with NMDG-Cl (100 mM) in place of NaCl in both the external and internal buffer was also carried out according to the above procedures, except that the POPC lipid concentration was increased to 0.1 mM and NMDG (20 µL of 0.5 M giving a final concentration 5 mM in the cuvette) was used for the base pulse in place of NaOH. This assay was carried out with and without the addition of the proton channel Gramicidin D (0.1 mol%, added in 4 μL DMSO), which was added 40 s before the base pulse .

HPTS assay data for transporters
In the following figures: Left: change in relative fluorescence intensity over time in the HPTS assay (LUVs (31.3 !M lipid); inside 100 mM NaCl, 10 mM HEPES, 1 mM HPTS, pH 7.0; outside: 100 mM NaCl, 10 mM HEPES, pH 7.0). Right: dependence of the fractional transport activity y in the HPTS assay on the concentration of transporter (black squares) and fitted to the Hill equation (red line).   The decrease in transport with NaGlu for all carriers indicated they do not facilitate sodium cation transport (H+ /Na+ antiport), and must therefore operate via an anion transport mechanism.
Membrane fluidity studies Figure S4. The change in relative fluorescence intensity over time in the HPTS assay utilizing DPPC LUVs at 25°C and 45°C. The temperature of the sample was controlled using a Peltier temperature controller. 2·Te 2CF3 was administered (arrow) at a concentration of 62.5 nM. Lack of transport in the gel phase at 25°C, and restoration above the phase transition temperature (41°C) at 45°C, is consistent with a mobile carrier transport mechanism.
Pre-incorporation studies

NMDG Assay
The following figure shows the dependence of the fractional transport activity y in the NMDG assay on the concentration of transporter, and the corresponding fit to the Hill equation (red and blue lines). This assay was carried out with and without the addition of Gramicidin D (0.1 mol%, 4 μL DMSO solution) added 40 s before the NMDG base pulse (20 µL of 0.5 M). Error bars represent 2 s.d.    In the case of 1·Te 2CF3 , 1·Te Ph , 1·Se 2CF3 , 2·Se 2CF3 , 1·Se Ph and 2·Se Ph no chemical shift perturbations were observed after 10 equivalents of TBACl. A representative example is shown in for 2·Se 2CF3 below.  Links to the fitting data for: 2·Te 2CF3 http://app.supramolecular.org/bindfit/view/42f41203-0462-4eda-8a2e-dda40fface67 2·Te Ph http://app.supramolecular.org/bindfit/view/9f08d026-d73e-4a04-865b-365710abe1ad

NMR Switching Studies
All switching studies were carried with at concentration of 5 mM of diaryl compound.
1·Se 2CF3 [3] was prepared according to a literature procedure.
1·Se Ph was purchased from Sigma-Aldrich.
2·Se Ph was prepared according to a literature procedure. [2] S1 was prepared according to a literature procedure. [4] Tellurones, such as 3·Te 2CF3 and 3·Te Ph , present certain challenges with regards to their characterisation by NMR spectroscopy. The dynamic formation of higher aggregates and relatively poor solubility in most organic solvents causes significant broadening in 1 H, 13 C, 19 F and 125 Te NMR signals such that they are unobservable. It is important to recognise that this is a widely acknowledged characteristic of tellurones compounds. We present here the 1 H NMR spectra of 3·Te 2CF3 and 3·Te Ph to demonstrate this, but in all cases the [M+H] + cations were observable by ESI-MS. [5] Scheme S1. Synthetic route for the tellurium-based receptors.

General procedure 1
The telluride (10.0 mmol) was dissolved in 20 mL of 1/1 (v/v) methanol/CH2Cl2, and the solution was cooled to 0 °C. NCS (1.41 g, 10.5 mmol) was added, and the resulting solution was stirred for 30 min at 0 °C. The reaction mixture was diluted with 20 mL of methylene chloride, and 30 mL of a 10% sodium hydroxide solution was added. After the mixture was stirred for 5 min, the organic phase was separated, dried over MgSO4 and concentrated to dryness to afford the corresponding telluroxide as a white solid.

General procedure 2
The telluride (1.00 mmol) in ethanol (10 ml) was added a solution of sodium periodate (480.5 mg, 2.25 mmol) in water (10 ml). After being stirred at room temperature overnight, the mixture was diluted with water and partition with CHCl3 (500 ml), the organic phase was collected and dried over MgSO4 and concentrated to dryness affording the tellurone as a white solid.
Scheme S2. Synthetic route for the selenium based receptors.

General procedure 3
The selenide (10.0 mmol) was dissolved in 20 mL of CH2Cl2, and the solution was cooled to 0 °C. Br2 (10.5 mmol) was added, and the resulting solution was stirred for 30 min. The reaction mixture was diluted with 30 mL of a 10% sodium hydroxide solution was added. After the mixture was stirred for 5 min, the organic phase was separated, dried over MgSO4 and concentrated to dryness to afford the corresponding selenoxide as a white solid.

1·Te 2CF3
A solution of S1 (10 mmol) in anhydrous THF(50 ml) was cooled to 0°C. To which was added dropwise a 1 M Br2 solution in ether (10 mmol) and allowed to stir for 5 minutes, after which time 3,5-Bis(trifluoromethyl)phenyl magnesium bromide (11 mmol) was added dropwise. After 30 minutes of stirring at room temperature the reaction mixture was quenched by the addition of MeOH (10 ml) and the mixture concentrated in vacuo. The crude mixture was portioned between brine (250 ml) and CH2Cl2 (250 ml), concentrated to dryness and purified by silica gel column chromatography eluting with (CH2Cl2:hexane, 9:1, v/v) to afford 1·Te 2CF3 as orange solid (56%).

Single Crystal X-ray diffraction experiments
Crystal Structure Determination Deposition Numbers 2194578 (for 2·Te 2CF3 ) and 2194798 (for 2·Se 2CF3 ) contains the supplementary crystallographic data for this paper. These data are provided free of charge by the joint Cambridge Crystallographic Data Centre and Fachinformationszentrum Karlsruhe Access Structures service www.ccdc.cam.ac.uk/structures. Single-crystal X-ray diffraction intensities for 2·Te 2CF3 and 2·Se 2CF3 were collected at 150 K on Oxford Diffraction/Agilent SuperNovae diffractometers with Cu-Kα (λ = 1.54184 Å) radiation equipped with a nitrogen gas Oxford Cryosystems Cryostream unit A suitable crystal was chosen and mounted on a 200 μm MiTeGen loop using perfluoropolyether oil. The CrysAlisPro software was used for data collection and integration. Structure 2·Te 2CF3 was solved using SuperFlip and refined using full-matrix least-squares refinement within the CRYSTALS suite. Structure 2·Se 2CF3 was further refined using full-matrix least-squares refinement using Shelxl-2014. All non-hydrogen atoms were refined anisotropically. The hydrogen atoms were positioned at geometrically sensible positions and refined using a riding model. The compound crystallised with disordered solvent, which was treated using PLATON SQUEEZE. Disorder can also be observed for some of the CF3 groups, which has been modelled. However, some of the ADPs for the CF3 groups remain enlarged due to the disorder present.